In DUI animals, two sex-specific mitochondrial lineages are present: one transmitted maternally (F-type) and the other paternally (M-type), each containing a sex-specific mitochondrial ORFan gene. Comprehensive in silico characterization of DUI ORFan proteins has been conducted in freshwater mussels, with partial studies in marine mussels; however, research in other DUI species remains scarce. In this regard, we newly identified two DUI species, Antigona lamellaris (Schumacher, 1817) and Ezocallista brevisiphonata (P. P. Carpenter, 1864), and analyzed the major unassigned regions of the mitochondrial genomes in 169 specimens from eight species of Veneridae, verifying the presence of novel mitochondrial ORFs. Structural and functional analyses were performed on 14 ORFans, and the conservation patterns and properties of DUI ORFans in Veneridae were further explored by integrating in silico characterization results from freshwater and marine mussels. Strong structural and functional similarities were found between F-ORF and M-ORF proteins, both across different bivalve species and within the studied species. In contrast to freshwater and marine mussels, the F and M-ORF sequences in Veneridae show remarkable similarity. Additionally, apart from sex-specific ORFs, reverse ORFs (ORFs encoded on the reverse strand) were identified in Antigona lamellaris. Predicted transmembrane (TMs) number and topology analysis suggest that ORFs in Veneridae exhibit lower conservation compared to those in freshwater mussels. Our findings support the hypothesis that ORFans may originate from viruses or from proteins involved in mitochondrial energy production, providing further evidence for the multiple origins of ORFs. We propose that Veneridae, freshwater mussels, and marine mussels may harbor DUI systems with distinct origins and modes of action. The connection between ORFan proteins and DUI systems may differ across these taxa.

Genome research has dominated life sciences research in the last two decades. Of approximately 11,000 sequenced vertebrate genomes, genomes of teleost fish represent about 30%, with reference genome sequences available for most aquaculture fish species. While such progress has accelerated progress in aquaculture genetics research and breeding, it is clear that understanding of full genomic variations among aquaculture species is lacking. This is largely because of the way reference genomic sequences were produced, with a single or just a few genomes being sequenced. In addition, haplotype variations and their representation in the species or population are unknown. This hinders understanding of genomic basis of phenotypic variations relevant to performance and production traits such as growth rates, feed conversion efficiency, disease resistance, stress responses, processing yields, and reproductive traits, among other traits, especially so with strain-specific performance traits. The pangenome refers to a whole collection of genomic sequences found in the entire species or population rather than in a single individual, as represented in reference genomes. Pangenome includes the core genome sequence that are shared in all individuals, and variable or dispensable genome sequence found in a subset of individuals, representing intraspecies genomic variations. In this review, we present the current state of reference genomes and reference pangenomes, compare the advantages and disadvantages of various methods in producing pangenomes, propose the concept of pangenome plus (pangenome) to include genomes of related species with which interspecific hybrids can be made to introgress the beneficial genes. Revealing full genomic variations, determination of genomic variations relevant to performance traits, and combining beneficial genes and alleles into aquaculture breeds are three most important steps for the application of genome-based technologies to aquaculture breeding. To accomplish these three steps is challenging but offers unprecedented opportunities for aquaculture.

Developing effective molecular indicators to monitor stress status of coastal species is a top priority due to the impacts of climate change. However, the complexity of stress responses, which are regulated by multiple genes, limits the effectiveness of single-gene approaches in accurately reflecting stress status. Transcription factors (TFs) are promising candidates for comprehensively assessing stress responses, as they regulate numerous stress-responsive genes. In this study, we present a framework for identifying TF indicators that reflect the stress status of Pacific oysters (Crassostrea gigas). Specifically, oysters were exposed to high, medium, and low tide conditions to assess the physiological responses of oysters to tidal-induced stress. Enrichment analysis of differentially accessible chromatin peaks derived from assay for transposase-accessible chromatin sequencing (ATAC-seq) identified several key TFs. Among these, CCCTC-binding factor (Cg-CTCF) and MYB proto-oncogene A (Cg-MYBA) were significantly upregulated under tidal-induced stress and occupied critical positions in regulatory networks, as indicated by RNA-seq. RNA interference experiments confirmed that both genes contribute to enhancing survival under heat stress, a major stressor affecting oysters. Additionally, field experiments demonstrated significant upregulation of these genes under natural stress conditions, suggesting their potential as indicators for oyster reef management. To our knowledge, this is the first study on the coastal invertebrate to combine ATAC/RNA-seq with in vivo TF knockdown and field validation to propose TFs as practical stress indicators. We advocate for the broad application of our framework to explore TFs as molecular indicators of health status in marine organisms, thereby enabling informed strategies for conservation management.

Mollusk shell formation is a delicate and comprehensive physiological process that relies on the precise deposition of crystals under the control of shell matrix proteins. However, the underlying protective mechanisms governing this process remain largely unknown. Herein, a novel shell matrix protein gene was identified from the freshwater mussel, Hyriopsis cumingii. The predicted protein is characterized by a high glycine content and two Kunitz-type SPI domains, designated as HcGrKuSPI. The HcGrKuSPI gene was highly expressed in the outer epithelial of the mantle edge and mantle pallial. Immunostaining in situ analysis revealed the presence of HcGrKuSPI in the thick inter-prism matrix of the prismatic layer and the organic membrane of the nacreous layer. The expression of HcGrKuSPI in the pearl sac significantly increased during the ordered deposition of pearl nacre tablets. The resultant recombinant protein, SUMO-HcGrKuSPI, exhibited a strong affinity for aragonite and calcite and was involved in the morphological modification of calcite crystals in vitro. These results indicate that HcGrKuSPI is involved in organic framework construction and crystal morphological modification during the prismatic and nacreous layers formation. Furthermore, the expression of HcGrKuSPI in the mantle significantly increased following bacterial infection within the extrapallial fluid, and SUMO-HcGrKuSPI exhibited a strong inhibitory effect against bacterial growth and trypsin activity. Antibody injection in vivo led to severe damage to the thick inter-column framework of the prismatic layer and morphological deformities of nacre tablets. These findings indicate that HcGrKuSPI exhibits excellent protease inhibitory and antibacterial activities, providing a matrix protection system to ensure the smoothness of shell and pearl formation. Overall, these results enhance our understanding of the protective mechanisms involved in mollusk biomineralization.

Bivalves dominate global marine aquaculture, with shell biomineralization mediated by hemocytes and specialized proteins. Segon, the second-most abundant plasma protein in oysters, contains high calcium content and is hypothesized to participate in shell formation via calcium transportation. However, its gene family distribution and functional dynamics remain unexplored. This study aimed to systematically identify segon gene family, analyze their structural and evolutionary features, and elucidate expression patterns for developmental stages and shell damage response. Twelve segon genes were identified exclusively in family Ostreidae, with no gene found in other studied mollusks. All Segon proteins were predicted to be acidic (pI 4.98 ~ 6.07), hydrophilic, and localized to the extracellular space with a high Asp/Glu content indicating predicted calcium-binding capacity. Analysis of aligned segon sequences revealed that in Crassostrea virginica, segon and dominin did not form a fusion gene, whereas in all other species, segon sequences encompassed the dominin sequence. In silico predicted tertiary structures revealed conserved β-sheets and α-helices. segon transcript abundance was nearly undetectable in early larvae but sharply increased in spat-stage across oyster species, coinciding with the transition from aragonite to calcite shell polymorphs. The segon gene family is unique to Ostreidae and likely evolved through gene fusion events. Its spatiotemporal expression and structural features suggest that Segon possibly plays a vital role in calcium transport, offering novel targets for understanding oyster biomineralization and aquaculture enhancement.

The sulfated polysaccharides derived from marine species have attracted attention for their nutraceutical and pharmaceutical potential. Initially examined in cell culture, many labs have further tested their activity in in vivo rodent assays. Zebrafish embryos have been recently proposed as an alternative model species to test anti-inflammatory, fungicidal, immunomodulatory, antioxidant, antitumour, or toxicity effects with significant success. In this article, we systematically review by PRISMA strategy the assays that use zebrafish to evaluate potential nutraceutical and pharmaceutical applications of polysaccharides from more than 30 marine algal species. Effects against cancer, inflammation, and oxidative stress or over regeneration capacity, immunomodulation, and photoprotection in zebrafish are here compared. From this review, some polysaccharides appear as safe antitumour compounds (e.g. neutral S. skottsbergii-derived fraction over G-361 melanoma) while others show significant toxicity to embryos. The current bibliography shows a clear trend to use in parallel zebrafish larvae and in vitro assays to test the effects of polysaccharide fractions recovered from a variety of bioprocesses from different marine algae-derived biomass. Its potential application in the pharmacology and nutraceutical industry is discussed.

The study investigated the effect of ultrasonic radiation on the growth of Chlorella microalgae cells and the accumulation of intracellular metabolites (intracellular water-soluble proteins, lipids) in them. The results of the study showed that daily ultrasonic radiation (72 J/ml) with a power of 300 W for 60 s had a significant positive effect on the growth rate of microalgae. Increasing the ultrasound power from 20 W (4.8 J/mL) to 300 W (72 J/mL) resulted in a 1.3-fold increase in cell concentration in suspension and 1.4-fold increase in biomass concentration after 24 days of cultivation. The positive effect of ultrasound treatment on the process of periodic cultivation of microalgae is attributed to the acceleration of metabolism due to the increase in temperature of the suspension and the destruction of extracellular phenolic metabolites that inhibit the nitrate-anion transport system. In addition, ultrasound has a positive effect on the accumulation of intracellular metabolites in the biomass (the concentration of intracellular water-soluble proteins and total lipids became 1.4-fold and 2-fold higher, respectively, compared to the control). The research aims to increase the efficiency of the cultivation stage and may have practical significance for microalgae producers and researchers involved in the development of new methods to improve the production and obtain valuable substances from microalgae.

Perkinsus olseni is a highly pathogenic protozoan parasite affecting mollusks worldwide, posing a significant threat to Manila clam (Ruditapes philippinarum) aquaculture. This study investigated the impact of natural P. olseni infection abundance on the gill lipid metabolism of clams collected from Dandong City, Liaoning Province. The parasite species was identified via Polymerase Chain Reaction (PCR), while infection prevalence, abundance, and infection grade classification were assessed using Ray's Fluid Thioglycolate Medium (RFTM) culture. Lipid metabolomics analysis was performed to compare lipid profiles among uninfected (Neg), lightly infected (L), and moderately infected (M) clam gills. Results confirmed the presence of P. olseni with an overall prevalence of 91%. The infection abundance was significantly higher in the gills and digestive gland than in the mantle and siphon tissues. Lipidomic analysis of 18 samples (6 biological replicates per Neg, L, and M group) using a Liquid Chromatography-Mass Spectrometry (LC-MS) platform identified 1,561 metabolites, with significant differences observed across infection groups. 28 differential lipid metabolites were common to both the L and M groups when compared to the Neg group. KEGG pathway enrichment and correlation network analysis highlighted two key metabolites: diacylglycerol (DG) (16:0/18:2) and triacylglycerol (TG) (18:0/18:1/18:2). These metabolites were associated with 13 pathways, including T cell receptor signaling, fat digestion and absorption, and regulation of lipolysis in adipocytes. These findings elucidate tissue-specific infection patterns and reveal lipid metabolic reprogramming in clam gills in response to P. olseni, providing insights into host-parasite interactions and potential metabolic biomarkers for perkinsosis.

Copper ion pollution poses a significant threat to marine ecosystems. Seaweeds exhibit potential for metal bioremediation through their inherent mechanisms of metal absorption and tolerance. However, the understanding of seaweed resistance to metal ions, particularly copper, continues to evolve, despite previous studies elucidating some underlying molecular mechanisms. This study delineates the transcriptomic adaptations of Oocystis borgei to copper exposure via RNA-seq, highlighting its remarkable bioremoval efficacy, consistently averages 82%. RNA sequencing revealed profound alterations in transcriptomic profiles, which were further examined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses. This comprehensive examination identified key mechanisms facilitating copper absorption and enhanced tolerance in Oocystis borgei, including transporter activity, oxidation-reduction processes, photosynthesis, and glutathione metabolism. Overall, this analysis provides essential insights into the molecular mechanisms driving seaweed adaptive responses to copper-induced stress, laying the groundwork for future studies on their bioremediative capabilities.

At present, shells have been used to incubate Pyropia haitanensis (P. haitanensis) shell-boring conchocelis. To develop a more convenient substrate for P. haitanensis conchocelis cultivation, a novel P. haitanensis shell-boring conchocelis culture substrate (CaCO/LPE/CA) was developed from calcite, low-density polyethylene, and calcium alginate. The mechanical properties and stability of CaCO/LPE/CA, including implanting density (ID) and Yield per unit area, were tested. The results showed that the CaCO/LPE/CA met practical P. haitanensis shell-boring conchocelis breeding application requirements. P. haitanensis was successfully cultured on CaCO/LPE/CA, and its life cycle was observed through biomicroscopy and scanning electron microscopy (SEM). After conchospores was succrssfully reseased from P. haitanensis conchosporangial branch, the movement and germination of conchospores were also observed. The yield of conchospores per unit area of optimized CaCO/LPE/CA reached 369.6 × 10 conchospores/cm, making it a suitable candidate for P. haitanensis shell-boring conchocelis cultivation. Overall, CaCO/LPE/CA exhibits great potential as a marine bioactive material, and the process and cost of P. haitanensis culture breeding could be simplified due to its comprehensive characteristics.

Litopenaeus vannamei is one of the most widely farmed shrimp species worldwide, but it traditionally exhibits limited adaptability to low-salinity environments. Genetic improvement through intraspecific hybridization has been proven effective in enhancing environmental adaptability. This study aimed to elucidate the molecular mechanisms underlying the low-salinity adaptation of a hybrid shrimp strain selected through intraspecific hybridization. Shrimp were reared for 12 weeks under salinity conditions of 1 practical salinity unit (PSU) and 15 practical salinity units (PSU). By conducting a combined analysis of transcriptomics and metabolomics, we explored the low-salt adaptation mechanism of the hybrid shrimp. We found that they enhanced their adaptability through self-osmotic regulation and energy regulation. Transcriptome results revealed that genes associated with calcium-activated chloride channels, chloride transporters, and sodium-driven chloride/bicarbonate exchangers were up-regulated, suggesting enhanced ion transport capacity under low salinity. The metabolomics results indicated that key enzymes involved in glycolysis and gluconeogenesis, including phosphofructokinase and phosphoenolpyruvate carboxykinase, showed increased abundance, indicating elevated energy metabolism to support osmotic adjustment. Overall, the selected hybrid shrimp enhanced osmotic regulation by strengthening energy metabolism to improve their low-salt adaptability. These findings provide valuable insights for future genetic breeding and sustainable shrimp aquaculture in low-salinity regions.

China harbors extensive saline-alkaline water resources with considerable potential for aquaculture development. However, their utilization is constrained by high pH, elevated carbonate alkalinity, and complex ionic composition. Exopalaemon carinicauda, a commercially important shrimp species in China, is recognized for its environmental adaptability, rapid growth, desirable flesh quality, and high economic value. Owing to its resilience to diverse environments, E. carinicauda serves as an ideal model for investigating the molecular mechanisms underlying saline-alkaline adaptation in crustaceans. Yet, the genetic determinants of its alkalinity tolerance remain poorly understood, hindering selective breeding efforts. In this study, bulked segregant analysis (BSA) coupled with next-generation sequencing was employed to identify single nucleotide polymorphisms (SNPs) associated with alkalinity tolerance. DNA from individuals exhibiting extreme phenotypes was pooled, and allelic differences were assessed using Euclidean distance, deep learning, and ΔSNP-index methods. A total of 20,879,626 SNPs were detected, and seven candidate genomic regions spanning 47.53 Mb on chromosomes 4, 11, 13, 18, 26, and 36 were identified, encompassing 194 genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed significant associations with GABAergic synapse, taste transduction, and vasopressin-regulated water reabsorption. Comparative transcriptomic analysis under high-alkalinity stress in hepatopancreas and gills identified 28 genes strongly linked to alkalinity tolerance, including gamma-aminobutyric acid receptor, glutamate receptor ionotropic, and basic salivary proline-rich protein. Two SNP loci, C4-2601 and C8-6550, significantly associated with alkalinity tolerance were validated through PCR-based sequencing. These findings provide critical insights into the genetic architecture of alkalinity tolerance in E. carinicauda, facilitating future genomic and marker-assisted selection strategies.

Channa striata is a hardy fish that can thrive in various aquatic conditions; it is a good source of protein and has high economic value. This study was conducted to establish and characterize the muscle cell line of C. striata and use it to develop micro-tissue using seaweed biofilm. Explant culture was used to create a continuous muscle cell line from C. striata, which was then sub-cultured 143 times in Leibovitz L-15 medium with 10% FBS at 28 °C. They were properly cryopreserved, and after storage, 88-92% of the cells were recovered. The C. striata muscle cell line (CSM) was authenticated by examining the mitochondrial 16S rRNA gene using polymerase chain reaction. The immunophenotyping results revealed that the CSM cells were of myoblast origin, as evidenced by desmin and myosin cell markers. CSM cells showing a myogenic-like phenotype were also demonstrated through lipid droplets, which were confirmed by Nile Red staining. This cell line was devoid of mycoplasma infection. Inducing the foreign gene plasmid pEGFP-N1 resulted in a 16% transfection efficiency. Sterilized biocompatible seaweed biofilm was employed as a scaffold to grow micro-tissue using the developed CSM cell line. After 20 days of growth, this cell line produced micro-tissue on the seaweed biofilm. Seaweed biofilm is a one-of-a-kind biomaterial that has a wide range of biomedical applications, is ecologically friendly, biocompatible, and helps to improve cellular aquaculture and sustainable development.

Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.

Growth is an economically important traits in aquaculture. Previous studies identified ppp2ca as a candidate gene located within a major quantitative trait locus (QTL) for growth in Asian seabass. However, the molecular mechanisms underlying its role in growth regulation remain unclear. In this study, a single nucleotide polymorphism (SNP) in ppp2ca was found significantly associated with body weight. The gene was ubiquitously expressed across 11 examined tissues, with highest levels in the brain and eye, but reduced expression in the muscle of fast-growing fish. Functional assays showed that ppp2ca knockdown promoted cell proliferation, whereas its overexpression suppressed cell proliferation in an Asian seabass cell line. Two upstream mutations-a 10-bp indel and a 218-bp indel, were also identified and characterized. Dual-luciferase reporter assays demonstrated that the 10-bp insertion enhanced ppp2ca expression, while the 218-bp deletion reduced it. Permutation-based two-way ANOVA revealed a significant interaction between the 10 bp insertion and 218 bp deletion on body weight. These findings suggest that these upstream mutations in ppp2ca influence growth in Asian seabass, providing useful markers for selective breeding and key targets for future detailed functional studies on growth regulation.

The unreported peptide versicotide K (1), along with known averufin (2), 1'- hydroxyversicolorin B (3), averufanin (4), and sterigmatocystin (5), was isolated from the sponge-derived fungal strain Aspergillus versicolor 01NT- 1.5.1. Moreover, the new 6,8-dimethoxyaverythrin (6) and known averythrin (7) and sclerotiotide F (8) were isolated from another sponge-derived fungus Aspergillus flavus КMM 4695 (= VO49-48.3). The antimicrobial and cytotoxic activities of the isolated compounds were studied. The obtained data on the low cytotoxicity of averufanin (4) to normal HaCaT keratinocytes and H9c2 cardiomyocytes confirms its anticancer potential for future research. The significant activity of averythrin (7) against Staphylococcus aureus growth and biofilm formation (IC of approximately 10 µM) is the first. The anti-inflammatory activity of versicotide K (1) was predicted using the PASS online server. Moreover, SwissTargetPrediction services predicted COX2 as a possible target for 1, and the interaction of 1 with COX2 was calculated using a molecular docking approach. In in vitro experiments, versicotide K (1) reduced S. aureus infection and ischemia/reperfusion damage of H9c2 cells and prevented TNF-α induced damage of H9c2 cardiomyocytes by 24%, confirming its anti-inflammatory properties.

Light intensity, a crucial environmental factor, significantly affects the growth and physiological state of fish. This study investigated the effects of different light intensities (0, 150, 450, 650, and 1000 lx) on juvenile mandarin fish (Siniperca chuatsi) over a 66-day period. All groups achieved 100% survival, indicating that light intensity did not significantly affect survival. However, growth performance measured by specific growth rate (SGR) and weight gain rate (WGR) was highest at 150 lx. Nutritional analysis showed that crude protein and crude fat content were highest in the 150 lx group, while moisture was lowest under these conditions. Digestive enzyme activities in the liver, stomach, and intestine peaked under low-to-medium light intensities, with the highest values observed at 150 lx. Plasma cortisol levels were significantly lower at 150-450 lx than in other groups, indicating reduced stress under these conditions. In contrast, plasma melatonin levels declined with increasing light intensity and were lowest at 1000 lx. Overall, an illumination level of 150 lx was most beneficial for enhancing growth, reducing stress, and optimizing digestive enzyme activity in juvenile mandarin fish. These findings provide valuable insights for optimizing aquaculture practices and improving fish welfare and production efficiency.

Fish skeletal muscle serves as a crucial source of high-quality protein for human consumption. MicroRNAs (miRNAs) are important epigenetic regulators for the growth and development of skeletal muscle. Although the functions of many myogenic miRNAs have been studied, the regulatory functions of miRNAs in fish skeletal muscle have not been fully investigated. Here we show miR-125b is highly expressed in fast muscle of Chinese perch (Siniperca chuatsi) at 30-60 days post-hatching (dph), with transient downregulation during the skeletal muscle injury repair stage, implying its essential regulatory role in fast muscle growth and injury repair. Moreover, inhibiting miR-125b in Chinese perch resulted in an increase in muscle fiber diameter, the number of proliferating myoblasts and nuclei in single muscle fiber. In contrast, overexpression of miR-125b in Chinese perch led to a significant reduction in muscle fiber diameter, accompanied by a significant decrease in the number of proliferating myoblasts and the number of nuclei in single muscle fiber. Bioinformatics analysis and dual luciferase assays confirmed MyoD and Myomaker as direct targets of miR-125b. In summary, our findings demonstrate that miR-125b modulates the expression of MyoD and Myomaker, thereby regulating the proliferation and fusion of myoblasts, and ultimately controlling the hypertrophy of muscle fibers in Chinese perch. This finding holds significant relevance in unraveling the genetic mechanisms that govern the developmental traits of muscle fibers in fish during the postembryonic phase.

Antimicrobial resistance is a serious threat to global public health and requires new therapeutic approaches. Antimicrobial peptides (AMP) are recognized as promising candidates to address antimicrobial resistance. AMP can disrupt cell membranes by increasing permeability and causing lysis, or they can also interact with intracellular targets to inhibit essential metabolic processes. The genus Acropora is regarded as a valuable source for bioprospecting antimicrobial compounds. In this study, we employed in silico analytical strategies to predict potential antibacterial activity using AMPs derived from transcriptomes of multiple life cycle stages of the coral Acropora digitifera, as well as from cultured cells originating from adult coral tissues. The analysis involved multiple sequence alignments, Hidden Markov models, machine learning algorithms, structural modeling, physicochemical property assessment, and molecular docking. From the transcriptomic data, 15 sequences with potential antimicrobial activity were identified. Five AMPs were further evaluated for their binding efficacy against the TolC and OprM protein channels of RND-type transporter proteins, as well as DNA gyrase B of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Binding free energy analysis indicated that AMP-Ad2 exhibited the most favorable interaction with the TolC channel of E. coli. AMP-Ad3 showed the highest binding affinity with the OprM channel of P. aeruginosa, while AMP-Ad15 displayed the most favorable binding energy for the TolC channel of K. pneumoniae. The strongest interaction overall was observed between AMP-Ad15 and the DNA gyrase B of K. pneumoniae. These results demonstrate the utility of in silico prediction tools for identifying AMP candidates from A. digitifera transcriptomes and provide a basis for the planned synthesis and in vitro evaluation of these peptides, aiming to assess their therapeutic potential against resistant Gram-negative bacteria.

Tetrodotoxin (TTX), also known as puffer fish venom, has gained wide popularity in recent years as an effective tool in the field of physiology, as well as a promising anesthetic and analgesic agent. However, to date, no economically viable method of TTX production has been found. In this work, using high-performance liquid chromatography with tandem mass spectrometry, we conducted a study of the dynamics of the content and ratio of TTX and its analogues (TTXs) in the eggs of captive nemerteans of the Cephalothrix simula species complex, as well as a study of TTX localization in nemertean eggs using confocal laser scanning microscopy. It has been shown that nemertean eggs and nemerteans themselves, when kept in captivity for a long time, can contain concentrations of TTXs comparable to individuals of the wild population. It was found that TTX in eggs is associated with yolk granules, for which morphological characterization was carried out for the first time. The data obtained in the work can be used as a basis for the development of a technique for keeping animals in captivity for as long as possible while maintaining/increasing their toxic potential for further toxin extraction from egg yolk.